Epstein-Barr Virus (EBV) latent infections cause almost all EBV associated morbidity and mortality including lymphoblast proliferation early in Infectious Mononucleosis, Lymphoproliferative Diseases in people with AIDS and other immune compromised states, and EBV associated Lymphomas, Hodgkin's Disease, and Nasophryngeal Carcinoma. The EBV genome persists in all latently infected cells as a non-integrated multi-copy episome. The persistence of EBV episomes in dividing cells is dependent on the EBV encoded nuclear antigen 1 protein (EBNA1). EBNA1 binds to a specific site in the EBV episome and enhances episome initial replication, transcription, and persistence. Since EBNA1 is essential for the persistence of EBV episomes in all dividing and malignant cells, the central objective of this proposal is to identify compounds that can inhibit EBNA1 mediated episome persistence. To achieve that objective, we propose to: (1) Undertake screens to identify compounds that interrupt EBNA1-oriP dependent episome transcription and persistence in vivo, compounds that interrupt EBNA1 dimerization and binding to cognate DNA in vitro, and compounds that bind to EBNA1 in silico. (2) Identify the biological and biochemical effects of the identified compounds on EBNA1-oriP dependent episome transcription and persistence in B lymphoblasts, on EBV transformed lymphoblastoid cell (LCL) growth, and on LCL induced Lymphoma and NPC tumors in nude mice. (3) Determine the sites of bioactive compound effects in EBNA1 binding, using biochemical, biophysical, and structural approaches. Use this knowledge to most effectively undertake structure activity modifications to improve compound activity and specificity. (4) Use reverse genetics to identify the critical residues in EBNA1 DBD that can improve screening sensitivity and inform in silico pocket selection, compound modification, and compound interaction analyses.